A sapo peptide and a preparation method thereof

Abstract

The application discloses a small-molecule casein peptide and a preparation method thereof, and belongs to the field of small-molecule active peptides.The small-molecule casein peptide takes a small-molecule casein peptide chelating magnesium as an active ingredient, and the chelating rate of magnesium ions is greater than 70%.The preparation method comprises the following steps: adding pure water into casein powder and stirring to dissolve the casein powder; adding a protease for enzyme hydrolysis after adjusting pH and temperature; performing enzyme inactivation after the enzyme hydrolysis is completed; performing ultrafiltration separation on supernatant of the enzyme hydrolysis solution by using an ultrafiltration membrane to separate a casein peptide solution; adjusting the concentration of the casein peptide solution, and adding a magnesium-containing compound to perform a chelating reaction; adding anhydrous ethanol into the solution to precipitate the casein peptide after chelating; collecting the casein peptide precipitate after alcohol precipitation by centrifugation, and performing freeze-drying to obtain the small-molecule casein peptide chelating magnesium.The small-molecule casein peptide has significant physiological activity, good intelligence-improving effect, and wide application prospect; and the preparation method is simple, the reaction condition is mild, and the method is easy to be scaled up for industrial production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of small molecule active peptides, and particularly relates to a zizyphus jujuba casein peptide and a preparation method thereof. BACKGROUND

[0002] Casein is the main protein component in the milk of mammals including cows, sheep and humans. It is a large, hard, dense and extremely difficult to digest and decompose curd. The content of casein in cow's milk is about 80%, and the amino acid composition is relatively balanced, which can provide essential amino acids for the human body and support normal growth and development of the body. Studies have found that different enzymatic methods and processing conditions can obtain casein peptides with different functional characteristics. In recent years, the research on casein peptides has been continuously deepened, and it has been found that it has a variety of physiological functions, such as immune regulation, promotion of mineral absorption, antioxidant, antibacterial, blood pressure reduction, sleep promotion, etc.

[0003] With the development of society and the acceleration of people's life pace, the attention to brain health and intelligence improvement is increasing. Whether it is the student group in order to improve learning efficiency, or the job seekers in order to enhance work performance, there is a great demand for intelligence products, and various brain-nourishing health products and intelligence food are popular in the market and the market size continues to expand.

[0004] At present, the research on casein peptides in intelligence is relatively less, and only some studies show that casein peptides may have certain effects on the development and function of the nervous system, but its specific mechanism and effect still need to be further clarified.

[0005] In view of the potential intelligence function of casein peptides and the strong demand for intelligence products in the market, the development of an intelligence casein peptide has broad market prospects, not only meets the pursuit of consumers for safe and effective intelligence products, brings significant economic benefits to enterprises, but also helps to promote the development and innovation of the entire intelligence product industry, and leads to more research and application of functional peptides in the health field. SUMMARY

[0006] The purpose of the present application is to provide an intelligence casein peptide and a preparation method thereof. The intelligence casein peptide prepared by the present application has intelligence efficacy, filling the blank of casein peptides in intelligence.

[0007] The technical solution adopted by the present application to solve the technical problem is as follows:

[0008] The intelligence casein peptide provided by the present application takes small molecule casein peptides chelated with magnesium as the active ingredient.

[0009] As a preferred embodiment, the magnesium ion chelation rate of the intelligence casein peptide is > 70%.

[0010] The application provides a preparation method of a wisdom casein peptide, and specifically comprises the following steps.

[0011] (1) 5-20 times of pure water is added to casein powder, and the mixture is stirred and dissolved, and the pH is adjusted to 6-10 and the temperature is adjusted to 40-60 DEG C, and 0.3-1% of protease is added for enzymolysis for 3-6 h, and the enzyme is inactivated by increasing the temperature after the enzymolysis is completed;

[0012] (2) After the enzymolysis solution is centrifuged, the supernatant is obtained, and the supernatant is subjected to ultrafiltration treatment by using an ultrafiltration membrane to separate casein peptide solution;

[0013] (3) The concentration of the casein peptide solution is adjusted to 1-20%, and a magnesium-containing compound is added, and a chelation reaction is carried out at 35-45 DEG C and pH 6.5-7.5 for 1-5 h;

[0014] (4) Anhydrous ethanol is added to the solution after chelation, so that the final concentration of ethanol is 60-80%, and the solution is placed at 4 DEG C for 5-12 h, so that the casein peptide after chelation is precipitated and separated out;

[0015] (5) The casein peptide precipitate after alcohol precipitation is collected by centrifugation, and freeze-drying is carried out to obtain small molecule casein peptide chelated with magnesium.

[0016] As a preferred embodiment, in step (1), the enzyme is inactivated by increasing the temperature to 80-90 DEG C for 10-15 min after the enzymolysis is completed.

[0017] As a preferred embodiment, the protease is composed of bacillus subtilis protease and chymotrypsin, and the mass ratio of the bacillus subtilis protease and the chymotrypsin is (1-3):(1-3).

[0018] As a preferred embodiment, in step (2), the centrifugation condition is 5000-12000 rpm for 10-20 min.

[0019] As a preferred embodiment, in step (2), the molecular weight cut-off of the ultrafiltration membrane is 1000-2000 Da.

[0020] As a preferred embodiment, the magnesium-containing compound is selected from one of magnesium sulfate and magnesium chloride.

[0021] As a preferred embodiment, in step (3), the mass ratio of the casein peptide to the magnesium-containing compound is 1-10.

[0022] As a preferred embodiment, in step (3), the chelation reaction is carried out at a stirring speed of 200-600 rpm.

[0023] The application has the following beneficial effects:

[0024] 1. The casein peptide prepared by this invention has a magnesium-chelated small molecule casein peptide as its active ingredient. The magnesium ion chelation rate is >70%, and it has significant physiological activity, good brain-boosting effect, and broad application prospects.

[0025] 2. The method for preparing a brain-boosting casein peptide according to the present invention has a simple preparation process, mild reaction conditions, and is easy to scale up and transform into industrial production. Detailed Implementation

[0026] In a first aspect, the present invention provides a brain-boosting casein peptide.

[0027] This invention provides a brain-boosting casein peptide, which uses magnesium-chelated small-molecule casein peptides as its active ingredient. The magnesium ion chelation rate of this brain-boosting casein peptide is >70%, and experiments have proven that it has brain-boosting effects.

[0028] Secondly, the present invention provides a method for preparing brain-boosting casein peptides.

[0029] The present invention provides a method for preparing a brain-boosting casein peptide, which specifically includes the following steps:

[0030] Step S1: Enzymatic hydrolysis;

[0031] First, add 5 to 20 times the mass of pure water to the casein powder, stir to dissolve, and then adjust the pH of the solution to 6-10 and the temperature to 40℃-60℃. Then, add 0.3%-1% of protease for enzymatic hydrolysis for 3-6 hours. After the enzymatic hydrolysis is completed, raise the temperature of the solution to 80℃-90℃ (preferably 90℃) and keep it for 10-15 minutes (preferably 15 minutes) to inactivate the enzyme.

[0032] As a preferred embodiment, the method for preparing casein powder is as follows:

[0033] Fresh milk is degreased by centrifugation (45℃-50℃, 15000r / min) to remove fat to a fat content of ≤0.08%, followed by pasteurization (72℃, 15 seconds) to inactivate microorganisms. It is then concentrated three times by microfiltration using a 0.02μm stainless steel membrane at 3-5 bar pressure, with each concentration factor controlled at 4-7 times. Casein micelles are gradually separated using dilution filtration technology (water is added to half the original milk mass each time), resulting in a casein purity of over 80% in the final retentate. Vacuum concentration (50℃-70℃, vacuum degree 0.08MPa-0.09MPa) is then applied to a solids content of 30%-35%, followed by drying in a spray dryer (inlet temperature 120℃, outlet temperature 75℃) to form casein powder.

[0034] As a preferred embodiment, the protease consists of Bacillus subtilis protease and chymotrypsin, and the mass ratio of Bacillus subtilis protease and chymotrypsin is (1-3):(1-3).

[0035] Wherein, the Bacillus subtilis protease and chymotrypsin are all commercial products, for example, the Bacillus subtilis protease can be purchased from Dongheng Huadao, Novozymes, Lonza, etc.; the chymotrypsin can be purchased from Chongqing Jiangxia, Beijing Geyuan Tianlun, Sichuan Debol, etc.

[0036] Step S2: separation;

[0037] First, the enzyme solution obtained in step S1 is centrifuged at 5000 rpm-12000 rpm for 10 min-20 min to obtain the supernatant; then the obtained supernatant is subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da-2000 Da (preferably 1000 Da), and a casein peptide solution with a molecular weight of less than 1000 Da is separated.

[0038] Step S3: chelating magnesium;

[0039] First, the concentration of the casein peptide solution obtained in step S2 is adjusted to 1%-20%; then a magnesium-containing compound is added, wherein the mass ratio of casein peptide to magnesium-containing compound is 1-10, and the chelation reaction is carried out at a stirring speed of 200 rpm-600 rpm for 1 h-5 h under the conditions of temperature 35 ℃-45 ℃ and pH 6.5-7.5.

[0040] As a preferred embodiment, the magnesium-containing compound is selected from one of magnesium sulfate and magnesium chloride.

[0041] Step S4: alcohol precipitation;

[0042] First, anhydrous ethanol is slowly added to the chelated solution to make the final concentration of ethanol reach 60%-80%; then the chelated casein peptide is precipitated by standing at a temperature of 4 ℃ for 5 h-12 h.

[0043] Step S5: drying;

[0044] The casein peptide precipitate after alcohol precipitation is collected by centrifugation, and the chelated magnesium small molecule casein peptide is obtained after freeze-drying treatment.

[0045] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0046] Example 1 A method for preparing a casein peptide for improving intelligence

[0047] (1) Enzymolysis: First, 20 times the mass of pure water was added to the casein powder, and after stirring and dissolving, the pH of the solution was adjusted to 9.0 and the temperature was adjusted to 50°C; then 1% of a protease (the mass ratio of Bacillus subtilis protease: chymotrypsin was 2:1) was added for enzymolysis for 3 hours; after the enzymolysis was completed, the solution was heated to 90°C and maintained for 15 minutes to inactivate the enzyme.

[0048] (2) Separation: First, the enzymolysis solution obtained in step (1) was centrifuged at 10,000 rpm for 10 minutes to obtain the supernatant; then the obtained supernatant was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and a casein peptide solution with a molecular weight of less than 1000 Da was separated.

[0049] (3) Magnesium chelation: First, the concentration of the casein peptide solution obtained in step (2) was adjusted to 5%; then magnesium sulfate was added, and the mass ratio of casein peptide to magnesium sulfate was 1; under the conditions of a temperature of 40°C and a pH of 7.0, a chelation reaction was carried out at a stirring speed of 200 rpm for 4 hours.

[0050] (4) Alcohol precipitation: First, anhydrous ethanol was slowly added to the chelated solution to make the final concentration of ethanol 70%; then the chelated casein peptide was allowed to precipitate under the condition of a temperature of 4°C for 5 hours.

[0051] (5) Drying: The casein peptide precipitate after alcohol precipitation was collected by centrifugation, and after freeze-drying treatment, a small molecule casein peptide chelated with magnesium was obtained.

[0052] Example 2 A method for preparing a casein peptide for improving intelligence

[0053] (1) Enzymolysis: First, 10 times the mass of pure water was added to the casein powder, and after stirring and dissolving, the pH of the solution was adjusted to 8.0 and the temperature was adjusted to 45°C; then 0.8% of a protease (the mass ratio of Bacillus subtilis protease: chymotrypsin was 1:1) was added for enzymolysis for 4 hours; after the enzymolysis was completed, the solution was heated to 90°C and maintained for 15 minutes to inactivate the enzyme.

[0054] (2) Separation: First, the enzymolysis solution obtained in step (1) was centrifuged at 8000 rpm for 10 minutes to obtain the supernatant; then the obtained supernatant was subjected to ultrafiltration treatment using an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and a casein peptide solution with a molecular weight of less than 1000 Da was separated.

[0055] (3) Chelating magnesium: first, adjust the concentration of the casein peptide solution obtained in step (2) to 7%; then add magnesium chloride, and the mass ratio of casein peptide to magnesium chloride is 3, and the chelating reaction is carried out at a temperature of 38 ℃, a pH of 6.5 and a stirring speed of 300 rpm for 3 h.

[0056] (4) Alcohol precipitation: first, slowly add anhydrous ethanol to the chelated solution to make the final concentration of ethanol 80%; then, under the condition of a temperature of 4 ℃, stand for 7 h to make the chelated casein peptide precipitate.

[0057] (5) Drying: centrifugal collection of the casein peptide precipitate after alcohol precipitation, and the chelated magnesium small molecule casein peptide is obtained after freeze-drying treatment.

[0058] Test Example 1: Magnesium ion chelating rate test

[0059] Atomic absorption spectrometry (AAS) is used to determine the Mg 2+ concentration in the supernatant after alcohol precipitation in step (4) of Example 1 and Example 2, and the chelating rate is calculated by the following formula: chelating rate = (initial total Mg 2+ concentration - free Mg 2+ concentration in supernatant) / initial total Mg 2+ concentration) x 100%. Through the magnesium ion chelating rate test, the results show that the magnesium ion chelating rate of the casein peptide prepared by the present application is > 70%. Specifically as follows:

[0060] The magnesium ion chelating rate of the casein peptide obtained in Example 1 is measured to be 73.6%.

[0061] The magnesium ion chelating rate of the casein peptide obtained in Example 2 is measured to be 75.8%.

[0062] Test Example 2: Amino acid composition test of casein peptide prepared in Example 1 and Example 2 as test sample

[0063] After testing the amino acid composition of the casein peptides prepared in Example 1 and Example 2, the amino acid composition of the casein peptides is shown in Table 1 and Table 2. The casein peptides prepared in Example 1 and Example 2 contain various essential amino acids required by the human body, and the content of glutamic acid is more than 16%. Glutamic acid is closely related to learning, memory and cognitive function. In the learning process, glutamic acid can promote the change of synaptic plasticity, help the formation of new neural connections and strengthen memory, and also plays an important role in the normal maintenance and improvement of brain function.

[0064] Table 1: Test results of amino acid composition of casein peptide prepared in Example 1

[0065]

[0066] Table 2 Test results of amino acid composition of casein peptide prepared in Example 2

[0067]

[0068] Test Example 3: Intelligence function test using casein peptide prepared in Example 1 as a test sample

[0069] Experimental animals: 50 adult male mice, weighing 18-22 g, were randomly divided into 5 groups, 10 mice in each group.

[0070] Control group: normal mice; model group and low, medium and high dose groups: memory disorder model mice (intraperitoneal injection of scopolamine 5 mg / kg BW).

[0071] Low, medium and high dose groups and test sample administration time: the recommended oral dose of casein peptide for humans is 200 mg / day, and according to the standard body weight of adults of 60 kg, the equivalent dose is 3.34 mg / kg·bw. The experiment was divided into 5 groups (control group, model group, low dose group, medium dose group and high dose group), wherein the control group and the model group were given normal saline by gavage for 30 days, the low, medium and high dose groups were given the test sample by gavage for 30 days, the low dose group was fed according to the standard of 16.7 mg / kg·bw, the medium dose group was fed according to the standard of 33.4 mg / kg·bw, and the high dose group was fed according to the standard of 100.2 mg / kg·bw, which was about 5 times, 10 times and 30 times of the human oral dose (3.34 mg / kg·bw), respectively.

[0072] Test items and methods are as follows:

[0073] 1. Step test;

[0074] Training started the day after the last gavage. The mice were placed in the reaction box (on and under the platform) for 3 min to adapt to the environment, then the mice were placed on the copper grid in the reaction box, and immediately 36V alternating current was passed through. The normal response of the mice to the electric shock was to jump back to the platform (insulator) to avoid the harmful stimulus. Most mice may jump onto the copper grid again or multiple times, and quickly jump back to the platform after receiving the electric shock. Training for 3 min. 24 h later, the mice were placed on the platform, and the latency of the first jump off the platform of each mouse and the number of electric shocks within 3 min were recorded.

[0075] 2. Water maze test;

[0076] The pool was equally divided into 4 quadrants, and the platform was placed 2 cm below the horizontal plane of the 2nd quadrant. The middle positions of the 4 quadrants were used as the entry points, and the reference objects outside the maze remained unchanged during the training period. The mice were allowed to swim freely for 2 min to adapt to the environment 1 day before the experiment.

[0077] Morris water maze test includes two parts of place navigation experiment and spatial exploration experiment for 5 days. In the first 4 days of place navigation experiment, the mice are trained to find the platform 4 times a day. Each mouse enters the water from the four quadrants in the same order, and the time from entering the water to jumping on the platform is the escape latency. The results of the 4th day are used as the final results. On the 5th day, the platform is removed and the spatial exploration experiment is performed. A random entry point is selected, and the number of times and the effective time (the time of staying in the quadrant where the original platform is located) of the mouse crossing the original platform position within 2 min are recorded to reflect the spatial memory ability of the mouse to the platform position.

[0078] The test results are as follows:

[0079] 1. Step test;

[0080] The step test results are shown in Table 3. Compared with the control group, the latency of the mice in the model group was significantly reduced, and the number of errors was significantly increased. Compared with the model group, the latency of the mice in the low, medium and high dose groups was significantly prolonged, and the number of errors was reduced, indicating that casein peptide has a significant improvement effect on memory impaired mice, and the improvement effect is better with the increase of the dose.

[0081] Table 3 Effect of casein peptide on learning of mice in step test ±S, n=10

[0082]

[0083] 2. Water maze experiment;

[0084] The water maze experiment results are shown in Table 3. In the place navigation experiment, the mice in the model group learned more slowly than the mice in the control group. Compared with the model group, the escape latency of the mice in the low, medium and high dose groups was significantly reduced, and the effect of the mice in the high dose group was the most obvious. In the spatial exploration experiment, the number of times and the effective time of the mice in the model group crossing the platform were significantly reduced compared with the control group. Compared with the model group, the number of times and the effective time of the mice in the low, medium and high dose groups crossing the platform were significantly improved, and the effect of the mice in the high dose group was the best, indicating that casein peptide has a significant improvement effect on memory impaired mice, and the improvement effect is better with the increase of the dose.

[0085] Table 4 Effect of casein peptide on learning of mice in water maze experiment ±S, n=10

[0086]

[0087] Example 4: The casein peptide prepared in Example 2 was used as a test sample to test the intelligence function

[0088] The magnesium-chelated casein peptides obtained in Example 2 were tested for their effects on memory-impaired mice by the same method as in Test Example 3, and the results are shown in Table 5 and Table 6, showing the same improving effects as the magnesium-chelated casein peptides of Example 1.

[0089] Table 5 Effects of casein peptides on learning in the step-down test of mice ±S, n=10

[0090]

[0091] Table 6 Effects of casein peptides on learning in the water maze test of mice ±S, n=10

[0092]

[0093] In summary, the casein peptides prepared in the present application have good intelligence-improving effects.

[0094] The present application discloses an intelligence-improving casein peptide and a preparation method thereof, and those skilled in the art can refer to the content of the present application and appropriately improve the process parameters. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The product of the present application has been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the products described in the present application without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

Claims

1. A method for preparing a brain-boosting casein peptide, characterized in that, The brain-boosting casein peptide uses magnesium-chelated small molecule casein peptides as its active ingredient, and the magnesium ion chelation rate of the brain-boosting casein peptide is >70%. The preparation method includes the following steps: (1) Add 5-20 times the mass of pure water to casein powder, stir to dissolve, adjust pH to 6-10 and temperature to 40-60℃, add 0.3-1% protease for 3-6h enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, raise the temperature of the liquid to 80-90℃ and keep it for 10-15min to inactivate the enzyme. The protease is composed of Bacillus subtilis protease and chymotrypsin, and the mass ratio of Bacillus subtilis protease to chymotrypsin is (1-3):(1-3). (2) After centrifugation, the supernatant was obtained from the enzymatic hydrolysate. The supernatant was then subjected to ultrafiltration using an ultrafiltration membrane to separate casein peptide solutions with a molecular weight of less than 1000 Da. (3) Adjust the concentration of casein peptide solution to 1-20%, add magnesium-containing compound, and carry out chelation reaction for 1-5 h at 35-45℃ and pH 6.5-7.5; The magnesium-containing compound is selected from magnesium sulfate and magnesium chloride; The mass ratio of the casein peptide to the magnesium-containing compound is 1-10; (4) Add anhydrous ethanol to the chelated solution to make the final ethanol concentration 60-80%; let it stand at 4℃ for 5-12 hours to allow the chelated casein peptides to precipitate. (5) The casein peptide precipitate after alcohol precipitation was collected by centrifugation and then freeze-dried to obtain small casein peptides with chelated magnesium.

2. The method for preparing a brain-boosting casein peptide according to claim 1, characterized in that, In step (2), the centrifugation conditions are: 5000-12000 rpm, 10-20 min.

3. The method for preparing a brain-boosting casein peptide according to claim 1, characterized in that, In step (3), the chelation reaction is carried out with a stirring speed of 200-600 rpm.

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